Nitric oxide (NO) is a simple diatomic molecule that plays a diverse and complex role in cellular physiology. It is known that NO is a powerful signaling compound and cytotoxic/cytostatic agent found in nearly every tissue of the human body, including endothelial cells, neural cells, and macrophages. NO has been implicated recently in a variety of bioregulatory processes, including normal physiological control of blood pressure, angiogenesis, and thrombosis, as well as neurotransmission, cancer, and infectious diseases. See, e.g., Moncada, “Nitric Oxide,” J Hypertens. Suppl. 12(10): S35-39 (1994); Moncada et al., “Nitric Oxide from L-Arginine: A Bioregulatory System,” Excerpta Medica, International Congress Series 897 (Elsevier Science Publishers B.V.: Amsterdam, 1990); Marletta et al., “Unraveling the Biological Significance of Nitric Oxide,” Biofactors 2: 219-225 (1990); Ignarro, “Nitric Oxide. A Novel Signal Transduction Mechanism for Transcellular Communication,” Hypertension 16: 477-483 (1990); Hariawala et al., “Angiogenesis and the Heart: Therapeutic Implications,” J.R. Soc. Med. 90(6): 307-311 (1997); Granger et al., “Molecular and Cellular Basis of Myocardial Angiogenesis,” Cell. Mol. Biol. Res. 40(2): 81-85 (1994); Chiueh, “Neuroprotective Properties of Nitric Oxide,” Ann. N.Y. Acad. Sci. 890: 301-311 (1999); Wink et al., “The Role of Nitric Oxide Chemistry in Cancer Treatment,” Biochemistry (Moscow) 63(7): 802-809 (1998); Fang, F. C., “Perspectives Series: Host/Pathogen Interactions. Mechanisms of Nitric Oxide-Antimicrobial Activity,” J. Clin. Invest. 99(12): 2818-25 (1997); and Fang, F. C., “Nitric Oxide and Infection,” (Kluwer Academic/Plenum Publishers: New York, 1999).
Glyceryl trinitrate and sodium nitroprusside are two examples of vasodilators that currently enjoy widespread clinical use and whose pharmacological actions result from their metabolic conversion in situ to NO-releasing species. See, e.g., Ignarro et al., J. Pharmocol. Exp. Ther. 218: 739-749 (1981); Ignarro, Annu. Rev. Pharmacol. Toxicol. 30: 535-560 (1990); and Kruszyna et al., Chem. Res. Toxicol. 3: 71-76 (1990). In addition, other agents have been described in the literature which release NO spontaneously or following metabolic conversion of their parent or prodrug forms. See, e.g., Drago, ACS Adv. Chem. Ser. 36: 143-149 (1962); Longhi and Drago, Inorg. Chem. 2: 85 (1963); Schönafinger, “Heterocyclic NO prodrugs,” Farmaco 54(5): 316-320 (1999); Hou et al., “Current trends in the Development of Nitric Oxide Donors,” Curr. Pharm. Des. 5(6): 417-441 (1999); Muscara et al., “Nitric Oxide. V. Therapeutic Potential of Nitric Oxide Donors and Inhibitors,” Am. J. Physiol. 276(6, Pt. 1): G1313-1316 (1999); Maragos et al., “Complexes of NO with Nucleophiles as Agents for the Controlled Biological Release of Nitric Oxide. Vasorelaxant Effects,” J. Med. Chem. 34: 3242-3247 (1991); Fitzhugh et al., “Diazeniumdiolates: pro- and antioxidant applications of the ‘NONOates,’”Free Radic. Biol. Med. 28(10): 1463-1469 (2000); Saavedra et al., “Diazeniumdiolates (Formerly NONOates) in Cardiovascular Research and Potential Clinical Applications,” Nitric Oxide and the Cardiovascular System (Humana Press: Totowa, N.J., 2000); and Yamamoto et al., “Nitric oxide donors,” Proc. Soc. Exp. Biol. Med. 225(3): 200-206 (2000).
NO-donor compounds can exert powerful tumoricidal and cytostatic effects. Such effects are attributable to NO's ability to inhibit mitochondrial respiration and DNA synthesis in certain cell lines. In addition to these bioregulatory properties, NO may arrest cell migration. These effects are apparently not limited to NO-donor compounds as macrophages can also sustain high levels of endogenous NO production via enzymatic mechanisms. Similar inhibitory effects have also been observed in other cells. See, e.g., Hibbs et al., “Nitric Oxide: A Cytotoxic Activated Macrophage Effector Molecule,” Biochem. and Biophys. Res. Comm. 157: 87-94 (1988); Stuehr et al., “Nitric Oxide. A Macrophage Product Responsible for Cytostasis and Respiratory Inhibition in Tumor Target Cells,” J. Exp. Med. 169: 1543-1555 (1989); Zingarelli, et al., “Oxidation, Tyrosine Nitration and Cytostasis Induction in the Absence of Inducible Nitric Oxide Synthase,” Int. J. Mol. Med. 1(5): 787-795 (1998); Yamashita et al., “Nitric Oxide is an Effector Molecule in Inhibition of Tumor Cell Growth by rIFN-gamma-activated Rat Neutrophils,” Int. J. Cancer 71(2): 223-230 (1997); Garg et al., “Nitric oxide-Generating Vasodilators Inhibit Mitogenesis and Proliferation of BALB/C3 T3 Fibroblasts by a Cyclic GMP-Independent Mechanisms,” Biochem. Biophys. Res. Commun. 171: 474-479 (1990); and Sarkar et al., “Nitric Oxide Reversibly Inhibits the Migration of Cultured Vascular Smooth Muscle Cells,” Circ. Res. 78(2): 225-30 (1996).
Medical research is rapidly discovering a number of potential therapeutic applications for NO-releasing compounds/materials, particularly in the fields of vascular surgery and interventional cardiology. For example, fatty deposits may build up on the wall of an artery as plaque. Over time as additional material is added, the plaque thickens, dramatically narrowing the cross-sectional area of the vessel lumen in a process known as arteriosclerosis. Blood flow to the heart muscle is compromised resulting in symptoms ranging from intermittent chest pain to easy fatigability. In an effort to reduce such symptoms and improve blood flow, patients with this condition may opt to undergo a procedure known as coronary artery bypass grafting (CABG). In a typical CABG procedure, a portion of a vein is removed from the leg. Sections of the vein are then used to bypass the site(s) of plaque-induced coronary artery narrowing. CABG involves a major surgical procedure wherein the patient's chest is opened to facilitate the operation; as a result, it carries with it appreciable morbidity and mortality risks. However, bypassing the site(s) of greatest narrowing with a grafted vein substantially alleviates the chest pain and fatigue that are common in this condition while reducing the risk of acute arterial blockage. A less invasive and increasingly common procedure for treating plaque-narrowed coronary arteries is called percutaneous transluminal coronary angioplasty (PTCA) (also known as balloon angioplasty). In PTCA, a catheter is inserted into the femoral artery of the patient's leg and threaded through the circulatory system until the site of coronary vessel occlusion is reached. Once at the site, a balloon on the tip of the catheter is inflated which compresses the plaque against the wall of the vessel. The balloon is then deflated and the catheter removed. PTCA results in dramatic improvement in coronary blood flow as the cross-sectional area of the vessel lumen is increased substantially by this procedure. However, complications of this procedure may include thrombus formation at the site of PTCA-treatment, vessel rupture from overextension, or complete collapse of the vessel immediately following deflation of the balloon. These complications can lead to significant alterations in blood flow with resultant damage to the heart muscle.
A more general complication of angioplasty is restenosis, a complex multi-factorial process that is initiated when thrombocytes (platelets) migrate to the injury site and release mitogens into the injured endothelium. Clot formation or thrombogenesis occurs as activated thrombocytes and fibrin begin to aggregate and adhere to the compressed plaque on the vessel wall. Mitogen secretion also causes the layers of vascular smooth muscle cells below the site of injury (neointima) to over proliferate, resulting in an appreciable thickening of the injured vessel wall. Within six months of PTCA-treatment roughly 30 to 50% of patients exhibit significant or complete re-occlusion of the vessel.
Nitric oxide has recently been shown to dramatically reduce thrombocyte and fibrin aggregation/adhesion and smooth muscle cell hyperplasia while promoting endothelial cell growth (Cha et al., “Effects of Endothelial Cells and Mononuclear Leukocytes on Platelet Aggregation,” Haematologia (Budap) 30(2): 97-106 (2000); Lowson et al., “The Effect of Nitric Oxide on Platelets When Delivered to the Cardiopulmonary Bypass Circuit,” Anest. Analg. 89(6): 1360-1365 (1999); Riddel et al., “Nitric Oxide and Platelet Aggregation,” Vitam. Horm. 57: 25-48 (1999); Gries et al., “Inhaled Nitric Oxide Inhibits Human Platelet Aggregation, P-selectin expression, and Fibrinogen Binding In Vitro and In Vivo,” Circulation 97(15): 1481-1487 (1998); and Lüscher, “Thrombocyte-vascular Wall Interaction and Coronary Heart Disease,” Schweiz. ‘Med. Wochenschr. 121(51-52): 1913-1922 (1991)). NO is one of several “drugs” under development by researchers as a potential treatment for the restenotic effects associated with intracoronary stent deployment. However, because the cascade of events leading to irreparable vessel damage can occur within seconds to minutes of stent deployment, it is essential that any anti-restenotic “drug” therapy be available at the instant of stent implantation. Also, it is widely thought that such therapy may need to continue for some time afterwards as the risk of thrombogenesis and restenosis persists until an endothelial lining has been restored at the site of injury.
In theory, one approach for treating such complications involves prophylactically supplying the PTCA-injury site with therapeutic levels of NO. This can be accomplished by stimulating the endogenous production of NO or using exogenous NO sources. Methods to regulate endogenous NO release have primarily focused on activation of enzymatic pathways with excess NO metabolic precursors like L-arginine and/or increasing the local expression of nitric oxide synthase (NOS) using gene therapy. U.S. Pat. Nos. 5,945,452, 5,891,459, and 5,428,070 describe the sustained NO elevation using orally administrated L-arginine and/or L-lysine while U.S. Pat. Nos. 5,268,465, 5,468,630, and 5,658,565 describe various gene therapy approaches. Other various gene therapy approaches have been described in the literature. See, e.g., Smith et al., “Gene Therapy for Restenosis,” Curr. Cardiol. Rep. 2(1): 13-23 (2000); Alexander et al., “Gene Transfer of Endothelial Nitric Oxide Synthase but not Cu/Zn Superoxide Dismutase restores Nitric Oxide Availability in the SHRSP,” Cardiovasc. Res. 47(3): 609-617 (2000); Channon et al., “Nitric Oxide Synthase in Atherosclerosis and Vascular Injury: Insights from Experimental Gene Therapy,” Arterioscler. Thromb. Vasc. Biol. 20(8): 1873-1881 (2000); Tanner et al., “Nitric Oxide Modulates Expression of Cell Cycle Regulatory Proteins: A Cytostatic Strategy for Inhibition of Human Vascular Smooth Muscle Cell Proliferation,” Circulation 101(16): 1982-1989 (2000); Kibbe et al., “Nitric Oxide Synthase Gene Therapy in Vascular Pathology,” Semin. Perinatol. 24(1): 51-54 (2000); Kibbe et al., “Inducible Nitric Oxide Synthase and Vascular Injury,” Cardiovasc. Res. 43(3): 650-657 (1999); Kibbe et al., “Nitric Oxide Synthase Gene Transfer to the Vessel Wall,” Curr. Opin. Nephrol. Hypertens. 8(1): 75-81 (1999); Vassalli et al., “Gene Therapy for Arterial Thrombosis,” Cardiovasc. Res. 35(3): 459-469 (1997); and Yla-Herttuala, “Vascular Gene Transfer,” Curr. Opin. Lipidol. 8(2): 72-76 (1997). However, these methods have not proved clinically effective in preventing restenosis. Similarly, regulating endogenously expressed NO using gene therapy techniques such as NOS vectors remains highly experimental. Also, there remain significant technical hurdles and safety concerns that must be overcome before site-specific NOS gene delivery will become a viable treatment modality.
The exogenous administration of gaseous nitric oxide is not feasible due to the highly toxic, short-lived, and relatively insoluble nature of NO in physiological buffers. As a result, the clinical use of gaseous NO is largely restricted to the treatment of neonates with conditions such as persistent pulmonary hypertension (Weinberger et al., “The Toxicology of Inhaled Nitric Oxide,” Toxicol. Sci. 59(1), 5-16 (2001); Kinsella et al., “Inhaled Nitric Oxide: Current and Future Uses in Neonates,” Semin. Perinatol. 24(6), 387-395 (2000); and Markewitz et al., “Inhaled Nitric Oxide in Adults with the Acute Respiratory Distress Syndrome,” Respir. Med. 94(11), 1023-1028 (2000)). Alternatively, however, the systemic delivery of exogenous NO with such prodrugs as nitroglycerin has long enjoyed widespread use in the medical management of angina pectoris or the “chest pain” associated with atherosclerotically narrowed coronary arteries. There are problems with the use of agents such as nitroglycerin. Because nitroglycerin requires a variety of enzymes and cofactors in order to release NO, repeated use of this agent over short intervals produces a diminishing therapeutic benefit. This phenomenon is called drug tolerance and results from the near or complete depletion of the enzymes/cofactors needed in the blood to efficiently convert nitroglycerin to a NO-releasing species. By contrast, if too much nitroglycerin is initially given to the patient, it can have devastating side effects including severe hypotension and free radical cell damage.
Because of problems associated with the systemic delivery of NO, there has been a recent shift towards identifying agents/materials capable of directly releasing NO or other antirestenotic agents over a prolonged period directly at the site of PTCA-vascular injury. As a result, there exists a substantial need for a stent comprised of or coated with a material capable of continuously releasing NO from the instant of contact with a blood field and subsequently releasing NO days or weeks following its deployment in a coronary artery. Such a device potentially represents an ideal means of treating the restenosis that frequently accompanies the implantation of a stent into a coronary artery. See, e.g., U.S. Pat. Nos. 6,087,479 and 5,650,447, U.S. Patent Application No. 2001/0000039, and PCT No. WO 00/02501, that detail approaches to develop NO-releasing coatings for metallic stents and other medical devices.
Diazeniumdiolates, compounds containing the moiety
comprise a diverse class of NO-releasing compounds/materials that are known to exhibit sufficient stability to be useful as therapeutics. Although the N-bound diazeniumdiolates were first discovered by Reilly (U.S. Pat. No. 3,153,094) and Drago et al., J. Am. Chem. Soc. 82: 96-98 (1960) more than 40 years ago, the chemistry and properties of these diazeniumdiolates have been extensively reinvestigated by Keefer and co-workers, as described in U.S. Pat. Nos. 4,954,526, 5,039,705, 5,155,137, 5,212,204, 5,250,550, 5,366,997, 5,405,919, 5,525,357, and 5,650,447, and in J. A. Hrabie et al., J. Org. Chem. 58: 1472-1476 (1993), and incorporated herein by reference.
Because many NO-releasing diazeniumdiolates have been prepared from amines, one potential approach for treating PTCA-associated restenosis is to coat the device with a suitably diazeniumdiolated amine-functionalized polymeric material. U.S. Pat. No. 5,405,919, for example, describes several biologically acceptable, amine-functionalized polyolefin-derived polymers. However, polyolefin-based coatings are prone to fractures as the coating is stressed during procedures such as stent expansion. Were such a fracture to occur, it might cause particulate fragments from the coating to be released into the lumen of the overstretched vessel, ultimately lodging downstream in much narrower arteriolae and capillaries and compromising blood flow to those portions of the heart muscle that are supplied by the affected artery. Additionally, polyolefin-based and -coated medical devices tend to be more prone to the development of biofilms and device-related infections. These problems suggest that polyolefin-based materials may not be appropriate for uses in which permanent in situ implantation is desired. By contrast, metallic medical devices have repeatedly been shown to exhibit bio- and hemocompatibility properties that are superior to many polyolefin-based materials. See, Palmaz, “Review of Polymeric Graft Materials for Endovascular Applications,” J. Vasc. Interv. Radiol. 9(1 Pt. 1): 7-13 (1998); Tepe et al., “Covered Stents for Prevention of Restenosis. Experimental and Clinical Results with Different Stent Designs,” Invest. Radiol. 31(4): 223-229 (1996); Fareed, “Current Trends in Antithrombotic Drug and Device Development,” Semin. Thromb. Hemost. 22(Suppl. 1): 3-8 (1996); Bolz et al., “Coating of Cardiovascular Stents with a Semiconductor to Improve Their Hemocompatibility,” Tex. Heart Inst. J. 23(2): 162-166 (1996); De Scheerder et al., “Biocompatibility of Polymer-Coated Oversized Metallic Stents Implanted in Normal Porcine Coronary Arteries,” Atherosclerosis 114(1): 105-114 (1995); and Libby et al., “Ultrasmooth Plastic to Prevent Stent Clogging,” Gastrointest. Endosc. 40(3): 386-387 (1994). More recently, quite dramatic improvements in bio- and hemocompatibility have also been observed in medical devices coated with certain polymeric materials (e.g., silicone, hydrogel, heparin-, albumin-, phosphorylcholine-functionalized polymers and the like). See, e.g., Malik et al., “Phosphorylcholine-Coated Stents in Porcine Coronary Arteries. In Vivo Assessment of Biocompatibility,” J. Invasive Cardiol. 13(3): 193-201 (2001); Tsang et al., “Silicone-Covered Metal Stents: An In Vitro Evaluation for Biofilm Formation and Patency,” Dig. Dis. Sci. 44(9): 1780-1785 (1999); Kuiper et al., “Phosphorylcholine-coated Metallic Stents in Rabbit Illiac and Porcine Coronary Arteries,” Scand. Cardiovasc. J. 32(5): 261-268 (1998); and McNair, “Using Hydrogel Polymers for Drug Delivery,” Med. Device Technol. 7(10): 16-22 (1996).
When exposed to hydrogen ion (i.e., proton) donors such as, for example, water or physiological fluids, most diazeniumdiolates bearing unshielded and unprotected [(NO)NO]− groups rapidly break down via clean first-order kinetic processes in which an initial surge of NO is followed by a steadily but diminishing (i.e., first-order) rate of release until the entire NO content of the material has been exhausted. For most diazeniumdiolated compounds, such processes are complete within minutes to a few hours of the initial NO burst.
Simple diazeniumdiolate ions have a characteristic absorbance maximum at or near 250 nm and are relatively stable under basic conditions. The amine/NO complexes release NO upon dissolution at physiological conditions, pH 7.4 and 37° C. Drago (Reactions of Nitrogen (II) Oxide. ACS Advances in Chemistry Series. 1962, 36, 143-149) described the reaction of nitric oxide with primary and secondary amines to form intermolecular salts, where one amine exists as a diazeniumdiolate with a negative charge and the other amine is protonated, giving it a positive charge. Hrabie et al. (New Nitric Oxide Releasing Zwitterions Derived from Polyamines. J. Org. Chem., 1993, 58, 1472-1476) studied the reaction of polyamines with NO to form intramolecular salts, zwitterions, which exhibit varying release rates of NO from around 1.3 min to 20 hours depending on the structure of the attached polyamine. Other monodiazeniumdiolated (poly)amine compounds have been prepared (see, for example, U.S. Pat. Nos. 5,731,305, 5,250,550, and 5,155,137), but the NO release data only indicated a clean first order nitric oxide release.
It was believed that in order to treat a disorder with nitric oxide over a period of time, two compounds would have to be co-administered-one compound with a quick release of NO and a second compound with an NO release rate several times longer than the first compound. This route would require that both compounds be approved by the FDA, an expensive and time-consuming endeavor. Alternatively, the same compound could be administered multiple times in order to provide a lasting treatment. However, this method increases the cost of treatment because of increased dosing and subjects the patient to increased exposure to any potential side effects.
Hrabie et al. (Adducts of piperazine with Nitric Oxide. Organic Preparations and Procedures International, 1999, 31(2), 189-192 and U.S. Pat. No. 5,721,365) examined adducts of the cyclic polyamine piperazine with nitric oxide. The bisdiazeniumdiolate of piperazine was reported to have a biphasic release of NO with an initial half-life of 2.3 minutes and a secondary half-life of 5.0 minutes. In fact due to the similarity in the initial and secondary release rates for the bisdiazeniumdiolate of piperazine, it was initially believed that the two release rates were identical. Because the profile of the biphasic release of NO from the bisdiazeniumdiolate of piperazine was on such a similar time scale, and because the second half-life of NO release was only 5.0 minutes, such a compound is not practical to use for conditions requiring a release of NO greater than a five-minute half-life. In addition, the use of piperazine diazeniumdiolate in pharmaceutical compositions is not desirable because of the potential toxicity of its possible nitrosopiperazine metabolite.
Previous research also discloses polydiazeniumdiolated polymers. Such NO-releasing polymers can have multiple phases of NO release. However, NO-releasing polymers often have kinetics that are too difficult to control and/or reproduce. The polymer structure, ionophore concentration, plasticizer content, physical shape and size of polymer mass, conformation, and placement of N2O2−groups can affect how much and when the NO is released. Moreover, the administration of NO-releasing polymers can be hindered by poor solubility and lack of structural homogeneity.
Therefore, it would be beneficial to administer a single, physiologically acceptable compound having a polyphasic NO release profile, where the initial first order release or burst of NO is on a relatively short time scale (e.g., seconds to minutes) and the additional first order release(s) of NO is on a longer relative time scale (e.g., hours to days). A compound capable of polyphasic release of NO would enable an acute treatment (e.g., the initial burst of NO) and sustained treatment (e.g., additional phase releases of NO) of a disorder in which the patient would receive the maximum benefits of treatment with a minimal dosage. Administration of a single compound with polyphasic NO release would require FDA approval of only one compound and would decrease the dosages and any potential side effects. Such compounds would additionally be beneficial to provide medical devices, e.g., stents, in the prevention of restenosis after removal of an arterial blockage, that are used in conjunction with such compounds as a means for effective delivery of the compounds.
The invention provides for such a compound. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.